Principal Investigator/ProgramDirector (Last, First, Middle): Ping, Peipei (MacLellan,Project 4) The theme of this Program Project application is to understand the signal transduction pathways mediating ischemic injury and cardioprotection using a multidisciplinary approach combining biophysics, physiology, proteomics and genetics. While Projects 1-3 are focused on two signaling pathways previously known to modulate ischemic injury and protection (i.e., PKCe and p38 MAPK), Project 4 focuses on a pathway with recently recognized importance in the field of myocardial ischemia[unreadable]cell cycle regulatory proteins, in particular, it will focus on three molecules whose functions are interrelated in this pathway: the cyclin-dependent kinase-2 (Cdk2), the retinoblastoma gene product (Rb), and the transcription factors (E2Fs) that regulate genes responsible for cell cycle entry including Cyclin A and E, the catalytic partners of Cdk2. Studies proposed in Project 4 are supported 1) by recent evidence demonstrating that ischemic injury to the heart are accompanied by the upregulation of a number of cell cycle regulatory proteins including Cdk2; and 2) by striking preliminary data demonstrating that infarct size was increased in Rb-null myocardium subjected to regional ischemic injury. Conversely, treatment with a Cdk2 inhibitor in vivo blocked the expected increase in Cdk2 activity with ischemic injury and led to a reduced infarct size formation. This suggests a critical role for Cdk2 in ischemic injury and a cardioprotective role of the Rb protein against injury. Although these observations are intriguing, as the cellular mechanisms underlying these effects are unknown. In collaboration with Projects 1-3 and the Cores, Project 4 proposes 3 Aims. Aim 1 will elucidate the mechanisms underlying Cdk2's ability to modulate ischemic injury; In collaboration with the Heart Biology Core, this aim will determine how Cdk2 modulates mitochondrial function and the cell death pathways; In collaboration with the Proteomic Core, Aim 1 will identify novel Cdk2 substrates. Aim 2 will examine the molecular basis forRb's cardioprotective role in ischemic injury, it will determine p38a MAPK dependent modulation of Rb, and will define the role of E2F family (theprimary targets of Rb) in these processes. Finally, Aim 3 will elucidate mechanisms by which PKCe regulates Cdk2 activity in cardioprotection. These studies will be performed in collaboration with Project 2 and the Heart Biology Core, and will employ two well-established murine models of cardioprotection: the PKCe transgenesis and the nitric oxide donor induced late phase of preconditioning. The proposed investigations will make key contributions to understanding the molecular basis for Rb's cardioprotective effect and the mechanism underlying Cdk2's ability to regulate ischemic damage and apoptotic cell death.